The long-term goal of the research proposal is to provide a thorough understanding of the space- and time-related changes associated with drug disappearance and metabolite formation in the liver, the major drug biotransformation organ. Methods for probing zonal metabolic heterogeneities: normal-retrograde (NR) perfusion and hepatic artery- portal vein, hepatic artery-hepatic vein (HAPV-HAHV) once-through rat liver perfusions, and the technique of multiple indicator dilution will be used to examine mechanisms of elimination and transport across hepatocyte membranes for several precursor-metabolite pairs. Following an injection of a mixture of vascular (51Cr-labeled red blood cells, 125I-labeled albumin, 58Co-EDTA which behaves similarly as 14C-sucrose) and cellular (D2O) markers and 14C- and 3H-labeled precursor and product during steady- state bulk perfusion by NR (portal or hepatic vein) and HAPV-HAHV (substrate and injection into hepatic artery), a set of indicator dilution outflow profiles are obtained. The physiological volumes, and the influx, efflux, and sequestration coefficients for precursor and metabolite may be obtained after appropriate modeling approaches. Specifically, the hepatic processing of acetaminophen-acetaminophen sulfate, salicylamide- salicylamide sulfate, enalapril-enalaprilat, morphine-morphine glucuronide, 4-methylumbelliferone-4-methylumbelliferyl conjugates and harmol-harmol conjugates (with and without deconjugation inhibitors, sodium sulfite, D- saccharo-1-4-lactone or D-glucarolactone) will be studied, as these represent simple (one step, two steps, and parallel) biotransformation pathways. Moreover, the role of the hepatic artery in hepatic drug and metabolite processing will be investigated with intravital microscopy, 14C- phenacetin and 3H-acetaminophen extraction data, and the technique of multiple indicator dilution, when the portal vein/hepatic artery flow ratios are altered. The results should lend important insights into mechanisms of drug-metabolite processing, the differential fates of metabolites, when generated intracellularly or when administered into the organ, due to the differing points of origin, reversible-futile metabolism, and on the role of the hepatic artery in hepatic metabolism. This work will also explain precursor-product relationships involved in drug- metabolite mediated toxicity/activity and improve the designs of prodrugs.